The present invention provides cell-based screening assays designed to identify agents that regulate the activity of the polycystic kidney disease proteins encoded by the PKD-1 and PKD-2 genes and that may be useful in the treatment of polycystic kidney disease. The assays of the invention comprise the contacting of genetically engineered cells expressing a mutant or truncated PKD gene product with a test agent and assaying for a decrease in the PKD-mediated mutant phenotype. Characteristics associated with such a mutant phenotype include increased adherence to type I collagen coated surfaces; apical expression of NaK-ATPase on the cell membrane; increased expression of xcex2-2-NaK-ATPase; and decreased focal adhesion kinase (FAK) incorporation into focal adhesion complexes, inability to form tubular structures in a gel matrix and decreased cell motility. To facilitate the screening methods of the invention, cells may be genetically engineered to express epitope tagged PKD gene products and/or epitope tagged PKD interacting proteins (PKD-IP). Such interacting proteins include, for example, focal adhesion complex proteins such as FAK, paxillin, vinculin, talin and the like.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common lethal genetic disease inherited as a dominant trait in humans, with a prevalence of 1:1,000 live births. The disease afflicts approximately 6 million people world-wide, and accounts for 5-7% of all patients on dialysis in the United States (Gabow, 1984, Ann. Intern. Med. 101:238-247). Mutations in the PKD-1 gene account for 85% of ADPKD, while mutations in the PKD-2 gene account for 10% of the disease. In both cases, ADPKD is characterized by progressive, massive cystic enlargement of renal tubules resulting from increased proliferation, aberrant secretion, altered membrane protein polarity, and extracellular matrix abnormalities correlated with a failure to down-regulate certain fetal genes after birth (Wilson, 1996, In Polycystic Kidney Disease, Oxford Clinical Nephrology Series, Watson M. L. and Torres V.E. eds. p. 124-163).
Approximately 50% of patients who inherit a mutant PKD-1 gene will develop end stage renal failure, typically in the 5 th decade of life, necessitating renal replacement therapy by dialysis or transplantation. Since progression is usually slow and is a consequence of gradual loss of renal function as cysts continue to enlarge and destroy intervening normal renal tubules, this presents a window of opportunity for potential drug therapies that would inhibit cyst expansion.
The PKD-1 gene maps to human chromosome 16p13.3, has 46 exons, encodes a 14.5 kb transcript with a 12,912 basepair open reading frame and translates into a 4303 amino acid (xe2x89xa7462 kDa) protein, referred to as xe2x80x9cpolycystin-1xe2x80x9d (European PKD Consortium, 1994, Cell 77:882-894; International PKD Consortium, 1995, Cell 81:289-298). The predicted amino acid sequence of the expressed protein suggests that the first 23 amino acids at the N-terminus act as a signal sequence, followed by two cysteine-flanked leucine rich repeats (LRR) which are strongly predictive of an extracellular location, protein-protein interactions and adhesion properties (Hughes et al., 1995, Nat Genet 10:151-160). Other putative extracellular domains include a C-lectin-like motif and a region with high homology to the receptor for egg jelly of sea urchins (REJ), implying potential calcium influx regulation (Moy et al., 1996, J. Cell. Biol. 133:809-817). The protein has several regions of high hydrophobicity and predicts 9-11 transmembrane domains and an intracellular carboxy terminal tail of 226 amino acids with putative binding sites for signal transduction molecules, including a SH2 site for tyrosine phosphorylation (YEMV) and two putative protein kinase C sites (RSSR) for serine phosphorylation.
The polycystin-1 protein is localized to areas of contact between the cell and matrix shortly after adhesion to type I collagen matrix. In addition, there is colocalization with defining focal adhesion proteins, namely xcex12xcex21-integrin, vinculin, xcex1-actin, talin, paxillin, focal adhesion kinase (pp 125FAK) and pp60c-src (Wilson et al., 1998, J. Cell. Biol. Vol. 9: 358A). Similar basally located polycystin-1-containing bodies have been demonstrated in vivo in human fetal ureteric bud epithelia in cell membrane regions in contact with type I collagen.
The overall predicted structure of the polycystin-1 protein and in vitro results with regard to matrix adhesion and phosphorylation assays, suggest that polycystin-1 functions as a matrix receptor mediating transfer of information from the extracellular matrix to the actin cytoskeleton, resulting in signal transduction that culminates in the nuclear regulation of gene transcription. This is suggested by the findings that all PKD-1 mutations mapped to date would predict the translation of a truncated protein product, lacking varying amounts of the C-terminal domain (CTD), including a potential SH2 site, and mutations that result in the failure to down regulate fetal expression of the xcex22 subunit of NaK-ATPase with consequent disruption of membrane polarization of NaK-ATPase (Peral et al., 1996, Am. J Hum. Gent. 58:86-96). Of additional interest, ADPKD epithelia have lower levels of PKD-1 tyrosine phosphorylation and fail to recruit FAK to the basally located multi-protein bodies.
In normal mature kidneys, NaK-ATPase is comprised of xcex11xcex21 heterodimers located at the basolateral membranes of renal tubules and is associated with vectorial Na+export into the basal cell space (blood side) driving ion gradients for fluid reabsorption. In normal fetal kidneys and also in ADPKD kidneys, the xcex22 subunit of NaK-ATPase heterodimerizes with al subunits which are xe2x80x9cmisxe2x80x9d targeted to apical plasma membranes, thus driving fluid secretion (Wilson et al., 1991, Am. J. Physiol. 260:F420-F430). The failure to repress xcex22 transcription in adult kidneys results, therefore, in fluid secretion and expansion of renal tubule lumens into cysts.
Less is known about the PKD-2 gene, which has been mapped to human chromosome 4q 21-23, encodes a 5.4 kb transcript and translates into a predicted 110kDa protein, xe2x80x9cpolycystin-2xe2x80x9d (Mochizuki et al., 1996, Science 272:1339-1342). Unlike polycystin-1, the PKD-2 encoded protein has intracellular C- and NH-termini, 6 transmembrane domains with a putative EF hand and coiled-coil domain in the C-terminal region and putative SH3 sites in the N-terminal region. Yeast 2 hybrid studies have suggested potential interactions of PKD-1 and PKD-2 coiled-coil domains and some co-localizations have been suggested, however, in strict contrast to polycystin-1, polycystin-2 is not developmentally regulated (Tsoikas et al., 1997, Proc. Natl. Acad. Sci. 94:6965-6970; Qian et al., 1997, Nature Genet. 16:179-183).
The present invention relates to cell-based screening assays designed to identify agents that regulate the activity of polycystic kidney disease proteins (encoded by the PKD-1 and PKD-2 genes). The assay system of the invention is based on the use of eukaryotic cells containing naturally occurring mutations in the PKD genes and/or eukaryotic cells genetically engineered to express different forms of the polycystic kidney disease genes (PKD genes) including wild type, mutant, truncated, or epitope tagged PKD encoded proteins. In specific nonlimiting examples of the invention, human renal cells are engineered to express mutant or truncated forms of PKD protein and express a mutant phenotype which includes one or more characteristics associated with renal epithelial cells from patients with polycystic kidney disease, for example, increased adherence to type I collagen coated surfaces; apical expression of NaK-ATPase on the cell membrane; increased expression of xcex2-2-NaK-ATPase; decreased FAK incorporation into focal adhesion complexes; and decreased cell motility, to name a few.
The assays provided by the present invention are designed to screen for compounds or compositions that modulate PKD activity, i.e., compounds or compositions that act as agonists or antagonists of PKD, and thereby regulate PKD mediated signal transduction and extracellular matrix interactions with the plasma membrane and cell cytoskeleton. The invention also relates to assays designed to screen for compounds or compositions that modulate PKD gene expression, i.e., compounds that modulate expression of PKD, or production or activity of transcription factors involved in PKD gene expression. Agents identified using the cell-based assays of the invention may be useful in the treatment of renal cystic disease.
In addition, the assays of the invention may be used to identify and/or validate protein interactions between PKD and binding partners within the cell, identified using different model host systems, i.e., C.elegans. To this end, cells endogenously or genetically engineered to express PKD may be further engineered to express PKD interacting proteins. Various standard assays may be used to validate such protein interactions and/or to serve as a basis for screening assays designed to identify agents that regulate the formation of such protein complexes.
As described herein, lithium chloride, a known activator of the wnt/xcex2-catenin signaling pathway, was shown to increase the motility of ADPKD cells to almost normal levels. Activation of the wnt/xcex2-catenin pathway is known to result in inhibition of serine/threonine protein kinase glycogen synthase kinase 3 (GSK-3). Thus the screening assays of the invention may be used to identify modulators of GSK-3 activity which can be used to regulate PKD mediated signal transduction.
As used herein, italicizing the name of a gene shall indicate the gene, in contrast to its encoded protein product which is indicated by the absence of italicizing.
For example, PKD shall mean the PKD gene, whereas PKD shall indicate the PKD encoded protein product. Unless otherwise specified, PKD shall refer to a PKD-1 or PKD-2 gene of any species, whereas PKD shall indicate an PKD-1 or PKD-2 encoded protein products (also referred to as polycystin-1 and polycystin-2, respectively) of any species. Such PKD genes and encoded proteins include, but are not limited to, those described by Mochizuki T et al. (1996, Science 272:1339-1342); The International Polycystic Kidney Disease Consortium (1995, Cell 81:289-298); and Barr and Sternberg (1999, Nature 401:386-389).